My laboratory is employing biochemical and genetic techniques to isolate and characterize mutations in hypoxanthine-guanine phosphoribosyltransferase in tissue culture cells. We have selected human and Chinese hamster cells lacking HGPRT activity with purine base analogs. To determine if the selected cells have altered HGPRT, we have purified and characterized Chinese hamster and human HGPRT and prepared monospecific antibody against both enzymes. Preliminary experiments indicate that antibody can be used to highly purify HGPRT protein from cell extracts. The purified mutant and wild-type proteins will be compared by peptide mapping. Tryptic and cynogen bromide peptides are being separated by size on sieving gels and by charge via high pressure ion-exchange chromatography. Radioisotope methods will be employed for the peptide mapping of the small amounts of enzyme protein obtainable from tissue culture cells. We can incorporate radioactivity into wild- type and mutant proteins either by labelling in vivo during cell growth with H3-amino acids, or by labelling the purified proteins after they have been isolated from the cells either with I125 or with C14- formaldehyde. We expect to identify the types and location of missense mutations in HGPRT. We hope to be able to identify a protein chain- termination mutation (amber or ochre mutation)) in HGPRT. If we find such a mutantion, we can search for revertants caused by a suppressor tRNA. Cells containing suppressor tRNAs would be of great value for the genetic analysis of oncogenic viruses. Bibliographic references: G. Milman, L.S. Portnoff, & D.C. Tiemeier. Immunochemical evidence for glutamine synthetase in cultured Chinese hamster cell. J. Biol. Chem. 250, 1393-1399 (1975). G. Milman, E.R. Ehnisz, A.S. Olsen, C.A. Maack, G.S. Ghangas, & S.A. Krauss. Analysis of hypoxanthine phosphoribosyltransferase mutant cell lines. Fed. Proc. 34, 588 (1975).